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- EMDB-9044: Yeast 26S proteasome bound to ubiquitinated substrate (5T motor state) -

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Entry
Database: EMDB / ID: 9044
TitleYeast 26S proteasome bound to ubiquitinated substrate (5T motor state)
Map dataYeast 26S proteasome bound to ubiquitinated substrate (5T motor state)
SampleSubstrate-engaged 26S proteasome in the 5T state
  • Proteasome
  • substrate
  • (Proteasome subunit alpha type- ...) x 6
  • Probable proteasome subunit alpha type-7
  • (26S proteasome regulatory subunit ...) x 5
  • 26S proteasome subunit RPT4
  • model substrate polypeptide
  • (ligand) x 2
Function / homologyNucleophile aminohydrolases, N-terminal / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome subunit / ATPase family associated with various cellular activities (AAA) / Proteasome subunit alpha 7-like / Proteasome alpha-subunit, N-terminal domain / Proteasome, subunit alpha/beta / AAA+ ATPase domain / ATPase, AAA-type, core ...Nucleophile aminohydrolases, N-terminal / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome subunit / ATPase family associated with various cellular activities (AAA) / Proteasome subunit alpha 7-like / Proteasome alpha-subunit, N-terminal domain / Proteasome, subunit alpha/beta / AAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / 26S proteasome regulatory subunit P45-like / Proteasome alpha-type subunit / AAA-protein family signature. / P-loop containing nucleoside triphosphate hydrolase / Proteasome subunit alpha 3 / Proteasome subunit alpha5 / Proteasome subunit alpha6 / Proteasome subunit alpha2 / Proteasome subunit alpha4 / Proteasome subunit alpha 1 / 26S Proteasome regulatory subunit 4 / 26S Proteasome regulatory subunit 7 / 26S Proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 8 / Proteasome alpha-type subunits signature. / 26S Proteasome regulatory subunit 6B / ABC-family proteins mediated transport / Antigen processing: Ubiquitination & Proteasome degradation / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / rt:r-sce-69229: / CDK-mediated phosphorylation and removal of Cdc6 / Orc1 removal from chromatin / Neutrophil degranulation / Ub-specific processing proteases / MAPK6/MAPK4 signaling / TNFR2 non-canonical NF-kB pathway / AUF1 (hnRNP D0) binds and destabilizes mRNA / SCF(Skp2)-mediated degradation of p27/p21 / Proteasome alpha-type subunit profile. / Cross-presentation of soluble exogenous antigens (endosomes) / SCF-beta-TrCP mediated degradation of Emi1 / protein-containing complex localization / proteasome regulatory particle assembly / nonfunctional rRNA decay / proteasome-activating ATPase activity / nuclear proteasome complex / proteasome regulatory particle, base subcomplex / cytosolic proteasome complex / peptide catabolic process / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / proteasomal ubiquitin-independent protein catabolic process / proteasome storage granule / proteasome core complex / proteasome endopeptidase complex / proteasome core complex, alpha-subunit complex / ubiquitin-dependent ERAD pathway / threonine-type endopeptidase activity / proteasomal protein catabolic process / TBP-class protein binding / positive regulation of transcription elongation from RNA polymerase II promoter / nucleotide-excision repair / positive regulation of RNA polymerase II transcriptional preinitiation complex assembly / positive regulation of protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of DNA-binding transcription factor activity / ubiquitin-dependent protein catabolic process / chromatin remodeling / endopeptidase activity / positive regulation of DNA-binding transcription factor activity / ATPase activity / mRNA binding / protein domain specific binding / mitochondrion / ATP binding / identical protein binding / nucleus / cytoplasm / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / 26S proteasome regulatory subunit 4 homolog / 26S proteasome subunit RPT4 / 26S proteasome regulatory subunit 8 homolog
Function and homology information
SourceSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 4.27 Å resolution
Authorsde la Pena AH / Goodall EA / Gates SN / Lander GC / Martin A
CitationJournal: Science / Year: 2018
Title: Substrate-engaged 26 proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.
Authors: Andres H de la Peña / Ellen A Goodall / Stephanie N Gates / Gabriel C Lander / A Martin
Abstract: The 26 proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and ...The 26 proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26 proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.
Validation ReportPDB-ID: 6ef2

SummaryFull reportAbout validation report
DateDeposition: Aug 15, 2018 / Header (metadata) release: Oct 17, 2018 / Map release: Oct 17, 2018 / Last update: Oct 17, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
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  • Surface view colored by radius
  • Surface level: 0.04
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  • Surface view with fitted model
  • Atomic models: : PDB-6ef2
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6ef2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9044.map.gz (map file in CCP4 format, 157217 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
340 pix
1.03 Å/pix.
= 350.2 Å
340 pix
1.03 Å/pix.
= 350.2 Å
340 pix
1.03 Å/pix.
= 350.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density
Contour Level:0.04 (by author), 0.04 (movie #1):
Minimum - Maximum0.0 - 0.19236124
Average (Standard dev.)0.0014019879 (0.007222254)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions340340340
Origin0.00.00.0
Limit339.0339.0339.0
Spacing340340340
CellA=B=C: 350.19998 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z350.200350.200350.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean0.0000.1920.001

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Supplemental data

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Mask #1

Fileemd_9044_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Mask #2

Fileemd_9044_msk_2.map
Projections & Slices
AxesZYX
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Slices (1/2)
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Mask #3

Fileemd_9044_msk_3.map
Projections & Slices
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Slices (1/2)
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Sample components

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Entire Substrate-engaged 26S proteasome in the 5T state

EntireName: Substrate-engaged 26S proteasome in the 5T state
Details: Yeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP
Number of components: 19

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Component #1: protein, Substrate-engaged 26S proteasome in the 5T state

ProteinName: Substrate-engaged 26S proteasome in the 5T state
Details: Yeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP
Recombinant expression: No

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Component #2: protein, Proteasome

ProteinName: Proteasome / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #3: protein, substrate

ProteinName: substrate / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria) / Strain: BL21

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Component #4: protein, Proteasome subunit alpha type-1

ProteinName: Proteasome subunit alpha type-1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.759469 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #5: protein, Proteasome subunit alpha type-2

ProteinName: Proteasome subunit alpha type-2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.07867 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #6: protein, Proteasome subunit alpha type-3

ProteinName: Proteasome subunit alpha type-3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.805178 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #7: protein, Proteasome subunit alpha type-4

ProteinName: Proteasome subunit alpha type-4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.862277 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #8: protein, Proteasome subunit alpha type-5

ProteinName: Proteasome subunit alpha type-5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.242596 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #9: protein, Proteasome subunit alpha type-6

ProteinName: Proteasome subunit alpha type-6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.355631 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #10: protein, Probable proteasome subunit alpha type-7

ProteinName: Probable proteasome subunit alpha type-7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.092719 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #11: protein, 26S proteasome regulatory subunit 7 homolog

ProteinName: 26S proteasome regulatory subunit 7 homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.452154 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #12: protein, 26S proteasome regulatory subunit 4 homolog

ProteinName: 26S proteasome regulatory subunit 4 homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.889051 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #13: protein, 26S proteasome regulatory subunit 8 homolog

ProteinName: 26S proteasome regulatory subunit 8 homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 29.010736 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #14: protein, 26S proteasome regulatory subunit 6B homolog

ProteinName: 26S proteasome regulatory subunit 6B homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 29.000164 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #15: protein, 26S proteasome subunit RPT4

ProteinName: 26S proteasome subunit RPT4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 29.353756 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #16: protein, 26S proteasome regulatory subunit 6A

ProteinName: 26S proteasome regulatory subunit 6A / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 29.845061 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae S288c (yeast)

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Component #17: protein, model substrate polypeptide

ProteinName: model substrate polypeptide / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 1.572701 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #18: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Component #19: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 25 mg/ml / pH: 7.6
Support filmunspecified
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 90 %
Details: specimens were manually blotted with Whatman #1 filter paper.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: images were acquired in nanoprobe mode
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1000.0 - -2500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 11656 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 47349 / Details: Camera was operated in counting mode
3D reconstructionSoftware: RELION
CTF correction: CTF correction was performed by Relion during reconstruction
Resolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 5MPC
Output model

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